Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild-type eyes, except Descemet's membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss-of-function mutations in collagen XVIII.     

The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa.

Type VII collagen is a major component of anchoring fibrils, attachment structures that mediate dermal-epidermal adherence in human skin. Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous disorder caused by mutations in the type VII collagen gene and perturbations in anchoring fibrils. In this study, we produced recombinant human type VII collagen in stably transfected human 293 cell clones and purified large quantities of the recombinant protein from culture media. The recombinant type VII collagen was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. Purified type VII collagen bound to fibronectin, laminin-5, type I collagen, and type IV collagen and also supported human dermal fibroblast adhesion. In an attempt to establish genotype-phenotype relationships, we generated two individual substitution mutations that have been associated with recessive DEB, R2008G and G2749R, and purified the recombinant mutant proteins. The G2749R mutation resulted in mutant type VII collagen with increased sensitivity to protease degradation and decreased ability to form trimers. The R2008G mutation caused the intracellular accumulation of type VII collagen. We conclude that structural and functional studies of in vitro generated type VII collagen mutant proteins will aid in correlating genetic mutations with the clinical phenotypes of DEB patients.     

Type VII collagen is a major structural component of anchoring fibrils

Anchoring fibrils are specialized fibrous structures found in the subbasal lamina underlying epithelia of several external tissues. Based upon their sensitivity to collagenase and the similarity in banding pattern to artificially created segment-long spacing crystallites (SLS) of collagens, several authors have suggested that anchoring fibrils are lateral aggregates of collagenous macromolecules. We recently reported the similarity in length and banding pattern of anchoring fibrils to type VII collagen SLS crystallites. We now report the construction and characterization of a murine monoclonal antibody specific for type VII collagen. The epitope identified by this antibody has been mapped to the carboxyl terminus of the major helical domain of this molecule. The presence of type VII collagen as detected by indirect immunofluorescence in a variety of tissues corresponds exactly with ultrastructural observations of anchoring fibrils. Ultrastructural immunolocalization of type VII collagen using a 5-nm colloidal gold-conjugated second antibody demonstrates metal deposition upon anchoring fibrils at both ends of these structures, as predicted by the location of the epitope on type VII collagen. Type VII collagen is synthesized by primary cultures of amniotic epithelial cells. It is also produced by KB cells (an epidermoid carcinoma cell line) and WISH (a transformed amniotic cell line).     

Type VII collagen forms an extended network of anchoring fibrils

Type VII collagen is one of the newly identified members of the collagen family. A variety of evidence, including ultrastructural immunolocalization, has previously shown that type VII collagen is a major structural component of anchoring fibrils, found immediately beneath the lamina densa of many epithelia. In the present study, ultrastructural immunolocalization with monoclonal and monospecific polyclonal antibodies to type VII collagen and with a monoclonal antibody to type IV collagen indicates that amorphous electron-dense structures which we term "anchoring plaques" are normal features of the basement membrane zone of skin and cornea. These plaques contain type IV collagen and the carboxyl-terminal domain of type VII collagen. Banded anchoring fibrils extend from both the lamina densa and from these plaques, and can be seen bridging the plaques with the lamina densa and with other anchoring plaques. These observations lead to the postulation of a multilayered network of anchoring fibrils and anchoring plaques which underlies the basal lamina of several anchoring fibril-containing tissues. This extended network is capable of entrapping a large number of banded collagen fibers, microfibrils, and other stromal matrix components. These observations support the hypothesis that anchoring fibrils provide additional adhesion of the lamina densa to its underlying stroma.     

The relationship of the biophysical and biochemical characteristics of type VII collagen to the function of anchoring fibrils

Type VII collagen is a major component of anchoring fibrils, which are 800-nm-long centrosymmetrically cross-banded fibrils that are believed to secure the attachment of certain epithelial basement membranes to the underlying stromal matrix. The ultrastructure of the anchoring fibrils is highly variable, suggesting that the fibrils are flexible. Flexibility measurements along the length of the triple-helical domain of type VII procollagen indicate that major flexible sites correlate well with known discontinuities in the (Gly-X-Y)n repeating sequence. Therefore, the helical disruptions may account for the tortuous shapes of anchoring fibrils observed ultrastructurally. The centrosymmetrical banding pattern observed for anchoring fibrils results from the unstaggered lateral packing of antiparallel type VII collagen dimers that form these structures. This antiparallel arrangement is specified by disulfide bonds formed at the margins of a 60-nm overlap of the amino termini. As long as these disulfide bonds remain intact, they protect the amino-terminal overlapping triple helices from collagenase digestion. This disulfide-bonded pair of triple helices is termed C-1. Large nonhelical domains (NC-1) extend from both ends of the anchoring fibrils and are believed to interact with the basement membrane or with anchoring plaques. Rotary shadowing of the NC-1 domains showed trident-like shapes, suggesting that a single alpha-chain contributed the structure of each arm and that the three arms were extended. Biochemical and biophysical analyses of NC-1 domains independently confirm these suggestions and imply that the arms of NC-1 domains are identical and individually capable of interactions with basement membrane components, potentially allowing trivalent interaction of type VII collagen with various macromolecules.     

Alpha 2 beta 1 integrin mediates dermal fibroblast attachment to type VII collagen via a 158-amino-acid segment of the NC1 domain.

Dermal fibroblasts are in apposition to type VII (anchoring fibril) collagen in both unwounded and wounded skin. The NC1 domain of type VII collagen contains multiple submodules with homology to known adhesive molecules, including fibronectin type III-like repeats and a potential RGD cell attachment site. We previously reported the structure and matrix binding properties of authentic and recombinant NC1. In this study, we examined the interaction between dermal fibroblasts and the NC1 domain of type VII collagen. We found that both recombinant and authentic NC1 vigorously promoted human fibroblast attachment. Adhesion of fibroblasts to NC1 was dose dependent, saturable, and abolished by both polyclonal and monoclonal antibodies to NC1. Cell adhesion to NC1 was divalent cation dependent and specifically inhibited by a monoclonal antibody directed against the alpha2 or beta1 integrin subunits, but not by the presence of RGD peptides. Furthermore, the cell-binding activity of NC1 was not conformation dependent, since heat-denatured NC1 still promoted cell adhesion. Using a series of recombinant NC1 deletion mutant proteins, the cell binding site of NC1 was mapped to a 158-aa (residues 202-360) subdomain. We conclude that human dermal fibroblasts interact with the NC1 domain of type VII collagen and this cell-matrix interaction is mediated by the alpha2beta1 integrin and is RGD independent.     

Supramolecular interactions in the dermo-epidermal junction zone: anchoring fibril-collagen VII tightly binds to banded collagen fibrils

The dermis and the epidermis of normal human skin are functionally separated by a basement membrane but, together, form a stable structural continuum. Anchoring fibrils reinforce this connection by insertion into the basement membrane and by intercalation with banded collagen fibrils of the papillary dermis. Structural abnormalities in collagen VII, the major molecular constituent of anchoring fibrils, lead to a congenital skin fragility condition, dystrophic epidermolysis bullosa, associated with skin blistering. Here, we characterized the molecular basis of the interactions between anchoring fibrils and banded collagen fibrils. Suprastructural fragments of the dermo-epidermal junction zone were generated by mechanical disruption and by separation with magnetic Immunobeads. Anchoring fibrils were tightly attached to banded collagen fibrils. In vitro binding studies demonstrated that a von Willebrand factor A-like motif in collagen VII was essential for binding of anchoring fibrils to reconstituted collagen I fibrils. Since collagen I and VII molecules reportedly undergo only weak interactions, the attachment of anchoring fibrils to collagen fibrils depends on supramolecular organization of their constituents. This complex is stabilized in situ and resists dissociation by strong denaturants.     

Epithelial origin of cutaneous anchoring fibrils

Anchoring fibrils are essential structural elements of the dermoepidermal junction and are crucial to its functional integrity. They are composed largely of type VII collagen, but their cellular origin has not yet been confirmed. In this study, we demonstrate that the anchoring fibrils are primarily a product of epidermal keratinocytes. Human keratinocyte sheets were transplanted to a nondermal connective tissue graft bed in athymic mice. De novo anchoring fibril formation was studied ultrastructurally by immunogold techniques using an antiserum specific for human type VII procollagen. At 2 d after grafting, type VII procollagen/collagen was localized both intracellularly within basal keratinocytes and extracellularly beneath the discontinuous basal lamina. Within 6 d, a subconfluent basal lamina had developed, and newly formed anchoring fibrils and anchoring plaques subjacent to the xenografts were labeled. Throughout the observation period of the experiment, the maturity, population density, and architectural complexity of anchoring fibrils beneath the human epidermal graft continuously increased. Identical findings were obtained using xenografts cultivated from cloned human keratinocytes, eliminating the possibility of contributions to anchoring fibril regeneration from residual human fibroblasts. Immunolabeling was not observed at the mouse dermoepidermal junction at any time. These results demonstrate that the type VII collagen of human cutaneous anchoring fibrils and plaques is secreted by keratinocytes and can traverse the epidermal basal lamina and that the fibril formation can occur in the absence of cells of human dermal origin.     

Differential Alterations in the Expression of Neurotransmitter Receptors in Inner Retina following Loss of Photoreceptors in rd1 Mouse

Loss of photoreceptors leads to significant remodeling in inner retina of rd1 mouse, a widely used model of retinal degeneration. Several morphological and physiological alterations occur in the second- and third-order retinal neurons. Synaptic activity in the excitatory bipolar cells and the predominantly inhibitory amacrine cells is enhanced. Retinal ganglion cells (RGCs) exhibit hyperactivity and aberrant spiking pattern, which adversely affects the quality of signals they can carry to the brain. To further understand the pathophysiology of retinal degeneration, and how it may lead to aberrant spiking in RGCs, we asked how loss of photoreceptors affects some of the neurotransmitter receptors in rd1 mouse. Using Western blotting, we measured the levels of several neurotransmitter receptors in adult rd1 mouse retina. We found significantly higher levels of AMPA, glycine and GABAa receptors, but lower levels of GABAc receptors in rd1 mouse than in wild-type. Since GABAa receptor is expressed in several retinal layers, we employed quantitative immunohistochemistry to measure GABAa receptor levels in specific retinal layers. We found that the levels of GABAa receptors in inner plexiform layer of wild-type and rd1 mice were similar, whereas those in outer plexiform layer and inner nuclear layer combined were higher in rd1 mouse. Specifically, we found that the number of GABAa-immunoreactive somas in the inner nuclear layer of rd1 mouse retina was significantly higher than in wild-type. These findings provide further insights into neurochemical remodeling in the inner retina of rd1 mouse, and how it might lead to oscillatory activity in RGCs.     

Cleavage of type VII collagen by interstitial collagenase and type IV collagenase (gelatinase) derived from human skin

Type VII collagen is the major structural protein of anchoring fibrils, which are believed to be critical for epidermal-dermal adhesion in the basement membrane zone of the skin. To elucidate possible mechanisms for the turnover of this protein, we examined the capacities of two proteases, human skin collagenase, which degrades interstitial collagens, and a protease with gelatinolytic and type IV collagenase activities, to cleave type VII collagen. At temperatures below the denaturation temperature, pepsin cleaves type VII collagen into products of approximately 95 and approximately 75 kDa. Human skin collagenase cleaved type VII collagen into two stable fragments of approximately 83 and approximately 80 kDa, and the type IV collagenase (gelatinase) produced a broad band of approximately 80 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleavage of type VII collagen was linear with time and enzyme concentration for both enzymes. Although the Km values were similar for both enzymes, the catalytic rate of cleavage by type IV collagenase is much faster than by interstitial collagenase, and shows a greater rate of increase with increasing temperature. Sequence analysis of the cleavage products from both enzymes showed typical collagenous sequences, indicating a relaxation in the helical part of the type VII collagen molecule at physiological temperature which makes it susceptible to gelatinolytic degradation. Interstitial collagenase from both normal skin cells and cells from patients with recessive dystrophic epidermolysis bullosa, a severe hereditary blistering disease in which both an anchoring fibril defect and excessive production of collagenase can be observed, produced identical cleavage products from type VII collagen. These data suggest a pathophysiological link between increased enzyme levels and the observed decrease or absence of anchoring fibrils.     

α2β1 Integrin Mediates Dermal Fibroblast Attachment to Type VII Collagen via a 158-Amino-Acid Segment of the NC1 Domain

Dermal fibroblasts are in apposition to type VII (anchoring fibril) collagen in both unwounded and wounded skin. The NC1 domain of type VII collagen contains multiple submodules with homology to known adhesive molecules, including fibronectin type III-like repeats and a potential RGD cell attachment site. We previously reported the structure and matrix binding properties of authentic and recombinant NC1. In this study, we examined the interaction between dermal fibroblasts and the NC1 domain of type VII collagen. We found that both recombinant and authentic NC1 vigorously promoted human fibroblast attachment. Adhesion of fibroblasts to NC1 was dose dependent, saturable, and abolished by both polyclonal and monoclonal antibodies to NC1. Cell adhesion to NC1 was divalent cation dependent and specifically inhibited by a monoclonal antibody directed against the α2 or β1 integrin subunits, but not by the presence of RGD peptides. Furthermore, the cell-binding activity of NC1 was not conformation dependent, since heat-denatured NC1 still promoted cell adhesion. Using a series of recombinant NC1 deletion mutant proteins, the cell binding site of NC1 was mapped to a 158-aa (residues 202–360) subdomain. We conclude that human dermal fibroblasts interact with the NC1 domain of type VII collagen and this cell–matrix interaction is mediated by the α2β1 integrin and is RGD independent.     

Human type VII collagen: genetic linkage of the gene (COL7A1) on chromosome 3 to dominant dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering disorders affecting the skin and the mucous membranes. Previous ultrastructural studies on the dystrophic (scarring) forms of EB have demonstrated abnormalities in the anchoring fibrils, morphologically distinct structures below the basal lamina at the dermal/epidermal basement membrane zone. Type VII collagen is the major collagenous component of the anchoring fibrils, and it is therefore a candidate gene for mutations in some families with dystrophic forms of EB. In this study, we performed genetic linkage analyses in a large kindred with dominant dystrophic EB. A 1.9-kb type VII collagen cDNA clone was used to identify a PvuII RFLP to follow the inheritance of the gene. This RFLP cosegregated with the EB phenotype in this family, strongly supporting genetic linkage (Z = 5.37; theta = .0). In addition, we assigned the type VII collagen gene (COL7A1) to chromosome 3 by hybridization to a panel of human x rodent somatic cell hybrids. These data demonstrate very close genetic linkage between the clinical phenotype in this family and the polymorphism in the type VII collagen gene mapped to chromosome 3. The absence of recombination between EB and the type VII collagen gene locus, as well as the observed abnormalities in the anchoring fibrils, strongly suggest that this collagen gene is the mutant locus in this kindred.     

Anchoring fibrils contain the carboxyl-terminal globular domain of type VII procollagen, but lack the amino-terminal globular domain

Type VII procollagen has been characterized as a product of epithelial cell lines. As secreted, it contains a large triple-helical domain terminated by a multi-globular-domained carboxyl terminus (NC-1), and a smaller amino-terminal globule (NC-2). The triple helix and the NC-1 domain have previously been identified in anchoring fibril-containing tissues by biochemical and immunochemical means, leading to the conclusion that type VII collagen is a major component of anchoring fibrils. In order to better characterize the tissue form of type VII collagen, we have produced a panel of monoclonal antibodies which recognize the NC-1 domain. Peptide mapping of these epitopes indicate that they are independent and span approximately 125,000 kDa of the total 150,000 kDa of each alpha chain contained in NC-1. All these antibodies elicit immunofluorescent staining of the basement membrane zone in tissues. Type VII collagen has been extracted from tissues. As previously reported, it is smaller than type VII procollagen, (Woodley, D. T., Burgeson, R. E., Lunstrum, G. P., Bruckner-Tuderman, L., and Briggaman, R. A., submitted for publication), and we now find that it predominantly occurs as a dimer. Following clostridial collagenase digestion, intact NC-1 has been recognized, indicating that the difference in apparent Mr between the tissue form of the molecule and type VII procollagen results from modification of the amino terminus. The size of the amino-terminal globule has been determined to be between approximately 96 and 102 kDa. Rotary shadowing analyses of extracted molecules indicate that dimeric molecules contain the NC-1 domain, but are missing intact NC-2. We propose that the tissue form monomer, Mr = 960,000, be referred to as "type VII collagen." These studies strongly suggest that anchoring fibrils contain dimeric molecules with intact NC-1 domains. The data also support the previous suggestion that the NC-2 domain is involved in the formation of disulfide bond-stabilized type VII collagen dimers, and is subsequently removed by physiological proteolytic processing.     

Hemidesmosomes and anchoring fibril collagen appear synchronously during development and wound healing

Bullous pemphigoid antisera and monoclonal antibodies to type VII collagen were used to localize hemidesmosomes and anchoring fibrils, respectively, in tissues of developing eyes and healing corneal wounds of New Zealand white rabbits. In the 17-day fetal rabbit eye, both antibodies colocalize to the epithelial-stromal junction of the lid and conjunctival region, but neither binds to the cornea, and electron microscopy demonstrates hemidesmosomes only where the antibodies bind. By 20 days of fetal development, the antibodies colocalize in cornea, and, by electron microscopy, hemidesmosomes are shown to be present as well. In healing 7-mm corneal wounds, both antibodies colocalize at the wound periphery within 66 h. By electron microscopy, hemidesmosomes along small segments of basal lamina are also shown to be present at the wound periphery at this time. These demonstrations of the synchronous assembly of hemidesmosomes and anchoring fibrils support the hypothesis of linkage of hemidesmosomes through the basement membrane to anchoring fibrils.     

The distribution of melanopsin (OPN4) protein in the human brain

Until now, melanopsin (OPN4) – a specialized photopigment being responsive especially to blue light wavelengths – has not been found in the human brain at protein level outside the retina. More specifically, OPN4 has only been found in about 2% of retinal ganglion cells (i.e. in intrinsically photosensitive retinal ganglion cells), and in a subtype of retinal cone-cells. Given that Allen Institute for Brain Science has described a wide distribution of OPN4 mRNA in two human brains, we aimed to investigate whether OPN4 is present in the human brain also at protein level. Western blotting and immunohistochemistry, as well as immunoelectron microscopy, were used to analyse the existence and distribution of OPN4 protein in 18 investigated areas of the human brain in samples obtained in forensic autopsies from 10 male subjects (54 ± 3.5 years). OPN4 protein expression was found in all subjects, and, furthermore, in 5 out of 10 subjects in all investigated brain areas localized in membranous compartments and cytoplasmic vesicles of neurons. To our opinion, the wide distribution of OPN4 in central areas of the human brain evokes a question whether ambient light has important straight targets in the human brain outside the retinohypothalamic tract (RHT). Further studies are, however, needed to investigate the putative physiological phototransductive actions of inborn OPN4 protein outside the RHT in the human brain.     

Molecular cloning and characterization of type VII collagen cDNA.

Type VII collagen, located in human epidermal basement membrane, is the primary pathogenic target molecule in epidermolysis bullosa acquisita and epidermolysis bullosa dystrophica. Using a monoclonal antibody against the non-collagenous domain of type VII collagen, approximately 1 Kb cDNA was isolated from human keratinocyte library. The deduced primary structure of this clone thus reflects the non-collagenous domain of type VII collagen that may be involved in cell attachment. This region shows a weak homology (approximately 23%) to the cell attachment domain of fibronectin. Northern blot revealed approximately 9.5 Kb single band.     

The occurrence of protein kinase C ϕ and λ isoforms in retina of different species

The localization and immunochemical identification of the novel protein kinase C ϕ (nPKC ϕ) and the atypical protein kinase C λ (aPKC λ) isoforms in retinas of different species were analyzed by immunohistochemistry and SDS-PAGE/Western blotting. nPKC ϕ immunoreactivity is associated with bipolar cells of mammalian (rabbit, rat and guinea pig) retinas but not the non-mammalian goldfish retina which has a lower concentration of nPKC ϕ. However, SDS-PAGE and Western blotting data indicate the antigen recognized by the nPKC ϕ monoclonal antibody in the retina is of a lower molecular weight than that expected for nPKC ϕ. This would suggest nPKC ϕ is more susceptible to degradation/breakdown than other PKC isoforms found in the retina or that the nPKC ϕ antibody may be recognizing an unknown retinal antigen. A comparison of nPKC ϕ and nPKC ϕ is present in the developing retina at an earlier stage than cPKC α. The typical ‘transport’ of cPKC α toward axonal terminals by phorbol-12,13-dibutyrate does not occur for nPKC ϕ yet both are translocated from the cytosolic to membrane compartments. The inner plexiform layer and the inner nuclear layer (putative horizontal cells) of all species examined (rabbit, rat, guinea pig and goldfish) exhibited positive immunoreactivity for aPKC λ as confirmed by SDS-PAGE/Western blotting. Special issue dedicated to Dr. Kinya Kuriyama.     

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.     

Astrocytes and Neurons Express the Tight Junction-Specific Protein Occludin in Vitro

The expression of occludin, an integral plasma membrane protein specifically located at tight junctions, was studied in various epithelial and nonepithelial tissues by means of RT-PCR, Western blotting, and immunofluorescent staining. Besides detection in epithelial and endothelial tissue, expression of occludin was found in primary and secondary cultures of neurons and astrocytes. Differentiation of astrocytes in vitro led to a marked decrease in occludin expression. Extractability of occludin from plasma membranes differed considerably between epithelial and nonepithelial cells. Following treatment with Triton X-100, occludin was completely extracted from astrocytic membranes but not from membranes derived from MDCK cells, suggesting a difference in the cytoplasmic and/or plasma membrane anchoring of occludin between these cell types.